U.S. patent application number 15/321726 was filed with the patent office on 2017-05-11 for microbial growth indicating medical devices.
This patent application is currently assigned to Indicator Systems International, Inc.. The applicant listed for this patent is Indicator Systems International, Inc.. Invention is credited to Robert M. Moriarty, Ram W. Sabnis, Gerald F. Swiss.
Application Number | 20170128595 15/321726 |
Document ID | / |
Family ID | 51867864 |
Filed Date | 2017-05-11 |
United States Patent
Application |
20170128595 |
Kind Code |
A1 |
Swiss; Gerald F. ; et
al. |
May 11, 2017 |
MICROBIAL GROWTH INDICATING MEDICAL DEVICES
Abstract
Provided herein are medical devices capable of self-reporting
microbial growth adjacent the site of the implanted device.
Inventors: |
Swiss; Gerald F.; (Rancho
Santa Fe, CA) ; Moriarty; Robert M.; (Michiana
Shores, MI) ; Sabnis; Ram W.; (Atlanta, GA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Indicator Systems International, Inc. |
Newport Beach |
CA |
US |
|
|
Assignee: |
Indicator Systems International,
Inc.
Newport Beach
CA
|
Family ID: |
51867864 |
Appl. No.: |
15/321726 |
Filed: |
June 22, 2015 |
PCT Filed: |
June 22, 2015 |
PCT NO: |
PCT/US2015/037031 |
371 Date: |
December 22, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14312541 |
Jun 23, 2014 |
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15321726 |
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PCT/US2014/037211 |
May 7, 2014 |
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14312541 |
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61821616 |
May 9, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61B 5/686 20130101;
A61B 5/412 20130101; A61K 49/0023 20130101; A61B 5/14539 20130101;
A61K 49/0043 20130101 |
International
Class: |
A61K 49/00 20060101
A61K049/00 |
Claims
1. A method of determining the presence of microbial growth at or
adjacent to a medical device placed on or in a patient which method
comprises: (a) selecting an implantable medical device having on at
least part of its surface self-identifying indicators which
indicators produce a differential signal under acidic pH as
compared to the signal produced at neutral or alkaline pH wherein
said signal can be assessed ex vivo; (b) placing said medical
device on or in a patient; (c) monitoring ex vivo the signal
produced by the self-identifying indicators; and (d) correlating
the signal so produced to the presence or absence of microbial
growth.
2. The method of claim 1, wherein said indicators are fluorescent
indicators which sense pH changes within physiological ranges.
3. The method of claim 1, further comprising: (e) measuring the
signal immediately after implantation to determine a first signal;
(f) measuring the signal at a later time to determine a second
signal; and (g) comparing the first signal and the second signal,
wherein a change in signal indicates the presence of the microbial
growth.
4. The method of claim 2, further comprising treating the patient
with one or more antimicrobial compounds if the presence of the
presence of microbial growth is determined.
5. A method to determine the microbe(s) present at or adjacent to a
medical device implanted in a patient which method comprises: (a)
selecting an implantable medical device having on at least part of
its surface self-identifying reporters which reporters produce a
differential signal when bound to a microbe as compared to the
signal produced when not bound to the microbe wherein said signal
can be assessed ex vivo, (b) placing said medical device in a
patient; (c) monitoring ex vivo the signal produced by the
self-identifying reports; and (d) correlating the signal so
produced to the presence or absence of the microbe at or adjacent
to the medical device implanted in the patient.
6. The method of claim 5, further comprising treating the patient
with one or more antimicrobial compounds if the presence of the
microbe is determined.
7. The method of claim 5, further comprising: (e) measuring the
signal immediately after implantation to determine a first signal;
(f) measuring the signal at a later time to determine a second
signal; and (g) comparing the first signal and the second signal,
wherein a change in signal indicates the presence of microbe.
8. The method of claim 7, further comprising treating the patient
with one or more antimicrobial compounds if the presence of the
microbe is determined.
9. A method of determining the presence of an infection at or
adjacent to a medical device placed on or in a patient which method
comprises: (a) selecting a medical device comprise self-identifying
indicators which indicators produce a differential signal under
acidic pH as compared to the signal produced at neutral or alkaline
pH wherein said signal can be assessed ex vivo; (b) placing said
medical device on or in a patient; (c) monitoring ex vivo the
signal produced by the self-identifying indicator; and (d)
correlating the signal so produced to the presence or absence of an
active infection.
10. The method of claim 9, wherein the medical device is a topical
device.
11. The method of claim 10, wherein said topical device is selected
from sutures, bandages, and wraps.
12. The method of claim 11, wherein said sutures are impregnated
with the self-identifying indicator.
13. The method of claim 12, wherein said self-identifying indicator
is a pH indicator or a pH sensitive fluorescent molecule.
14. The method of claim 13, wherein said self-identifying indicator
is fluorescein.
Description
FIELD OF THE INVENTION
[0001] This invention provides for the use of pH dependent
fluorescent molecules to determine the presence of an incipient
microbial infection in vivo. Such molecules can be used topically
in combination with wound dressings or in implantable medical
devices. In either case, these molecules are capable of
self-reporting microbial growth adjacent thereto. Further, the
invention provides for non-invasive methods to self-report the
microbes at a site having or suspected of microbial growth.
BACKGROUND OF THE INVENTION
[0002] Infections at the site of implantation of a medical device
are a serious problem. For example, surgeries relating to breast
implants result in infection rates from about 2% to as high as 20%
in women undergoing such implants, with the highest rate of
infection in reconstructive cases. Feldman, et al., Plast.
Reconstr. Surg., 126(3): 779-85 (2010). Similarly, prosthetic joint
infections are a frequent cause of prosthesis failure. Gemmel, et
al., Eur. J. Nucl. Med. Mol. Imaging, 39(5):892-909 (2012). A
variety of bacteria and fungi may be involved in such infections,
with staphylococci, including Staphylococcus epidermidis and S.
aureus, accounting for a majority of infections.
[0003] Frequently, infection of the site of implantation of a
medical device requires that the device be removed and/or replaced.
This results in increased risk to the patient as well as increased
cost. In addition, infection can lead to serious illness, and even
death, if the infection is unnoticed and untreated for even a
relatively short period of time. Undetected bacterial infection may
result in sepsis, septic phlebitis, septic shock, bacteraemia,
tunnel infection, and/or metastatic complications (e.g.,
endocarditis, osteomyelitis, or septic thrombosis). Accordingly,
early detection of bacterial infection in the region of the
implantation site of a medical device is highly desirable.
[0004] Therefore, a need exists for methods and medical devices for
the early detection of bacterial growth at or around the
implantation site of a medical device that can readily detect and
indicate the presence of microorganisms well before the infection
has progressed to the point that it manifests itself by clinical
symptoms.
SUMMARY OF THE INVENTION
[0005] This invention is related to the discovery that in vivo
microbial, such as bacterial, growth and infections, such as those
related to the implantation of a medical device will alter the pH
of the environment at and near the infection. Specifically,
physiologic fluid has a pH from about 7 to about 7.3. The presence
of an active microbial infection will result in production of
carbon dioxide and other components which, when mixed with
physiological fluid, convert to acidic components such as carbonic
acid. The presence of carbonic acid and other acidic components are
detectable by self-identifying indicators. These self-identifying
indicators produce a differential signal due to the pH change which
signal can be assessed ex vivo to ascertain the presence of an
infection in a patient.
[0006] Accordingly, in one embodiment, this invention is directed
to medical devices comprising on at least part of their surface
self-identifying indicators which indicators produce or can be
induced to produce a differential signal under acidic pH as
compared to the signal produced at neutral or alkaline pH wherein
said signal can be assessed ex vivo. The medical devices can
comprise implanted or topical devices such as artificial joints,
intravenous catheters, pace makers, sutures, wound coverings, and
the like.
[0007] In one embodiment, the self-identifying indicators can be
pH-dependent liposomes which comprise a paramagnetic ion under
neutral or alkaline pH. In another embodiment, these indicators are
pH sensitive dyes which change structure and hence alter at least
one of their electromagnetic emission characteristics in going from
an alkaline or neutral pH to an acidic pH. In yet another
embodiment, these indicators are pH sensitive fluorescent
indicators.
[0008] In one of its method aspects, this invention provides for an
ex vivo method to determine the presence of microbial growth at or
adjacent to a medical device implanted on or in a patient which
method comprises
[0009] selecting a medical device having on at least part of its
surface self-identifying indicators which indicators produce a
differential signal under acidic pH as compared to the signal
produced at neutral or alkaline pH wherein said signal can be
assessed ex vivo,
[0010] placing said medical device on or in a patient;
[0011] monitoring ex vivo the signal produced by the
self-identifying indicators; and
[0012] correlating the signal so produced to the presence or
absence of microbial growth.
[0013] In one embodiment, the medical devices contain
self-identifying reporters either by themselves or in combination
with the indicators set forth above. Such reporters include
compounds bound to the antibody or binding fragment thereof and
which emit a differential signal when bound to the microbe as
compared to that when not bound to the microbe. For example, such a
differential signal can be a signal arising from a change in at
least one electromagnetic emission character of the reporter when
bound as opposed to when not bound to the microbe.
[0014] In yet another embodiment, these reporters are fluorescent
indicators which have an altered fluorescence when bound to the
microbe as compared to being unbound.
[0015] In another embodiment, the medical device contains on at
least part of its surface an antibody or binding fragment thereof
which specifically binds to a microbe and produces a signal
indicating the identity of the microbe bound thereto. In some
embodiments, the antibody or binding fragment thereof has bound
thereto a reporter, such as a fluorescent moiety which changes its
fluorescent character upon binding to the microbe. In some
embodiments, a plurality of different antibodies or binding
fragments thereof are bound to the medical device, each producing a
unique signal for the microbe bound thereto.
[0016] Also provided herein are ex vivo methods to determine the
microbe present in an infection at or adjacent to a medical device
implanted in a patient which method comprises
[0017] selecting an implantable medical device having on at least
part of its surface self-identifying reporters which reporters
produce a differential signal when bound to a microbe as compared
to the signal produced when not bound to a microbe wherein said
signal can be assessed ex vivo,
[0018] placing said medical device in a patient;
[0019] monitoring ex vivo the signal produced by the
self-identifying reports; and
[0020] correlating the signal so produced to the presence or
absence of the microbe at an active infection.
[0021] In one embodiment, the signal produced by the indicator
and/or reporter on the implanted medical device is measured
immediately after implantation and that signal is used as a
baseline or reference signal for comparison to future signals so as
to aid the clinician in determining the degree of change in the
emitted signal or signals.
[0022] In some embodiments, the method further comprises treating a
patient with antimicrobial compounds, such as antibiotics.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 is the absorbance spectrum of heptamethoxy red at
neutral pH.
[0024] FIG. 2 is the absorbance spectrum of heptamethoxy red at an
acidic pH.
DETAILED DESCRIPTION OF THE INVENTION
[0025] This invention provides for medical devices capable of
self-identifying the presence of microbial growth at or adjacent
the medical device and/or the infecting microbe. However, prior to
discussing this invention in detail, the following terms will first
be defined.
DEFINITIONS
[0026] It must be noted that, as used in the specification and any
appended claims, the singular forms "a," "an," and "the" include
plural referents unless the context clearly dictates otherwise.
Similarly, the plural forms include singular referents unless the
context clearly dictates otherwise.
[0027] As used herein, the term "comprising" is intended to mean
that the compositions and methods include the recited elements, but
do not exclude others. "Consisting essentially of" when used to
define compositions and methods, shall mean excluding other
elements of any essential significance to the combination when used
for the intended purpose. Thus, a composition consisting
essentially of the elements as defined herein would not exclude
trace contaminants or inert carriers. "Consisting of" shall mean
excluding more than trace elements of other ingredients and
substantial method steps. Embodiments defined by each of these
transition terms are within the scope of this invention.
[0028] As used herein, an "implantable medical device" refers to
any type of medical device that is totally or partly introduced,
surgically or medically, into a patient's body or by medical
intervention into a natural orifice, temporarily or for a period of
time. In some aspects, the medical device is intended to be removed
during or upon completion of the procedure. In some aspects, the
medical device is intended to remain there after the procedure. The
duration of implantation may be between transient, such as for the
duration sufficient to retrieve a sample form the patient's body,
and essentially permanent, i.e., intended to remain in place for
the lifespan of the product or patient; or until it is physically
removed or biodegrades. Examples of essentially permanent
implantable medical devices include, without limitation,
implantable cardiac pacemakers and defibrillators; leads and
electrodes for the preceding; implantable organ stimulators such as
nerve, bladder, sphincter and diaphragm stimulators; cochlear
implants; prostheses, including artificial knees, hips, and other
joint replacements; vascular grafts, self-expandable stents,
balloon-expandable stents, stent-grafts, grafts, artificial heart
valves, cerebrospinal fluid shunts; renal dialysis shunts;
artificial hearts; implantable infusion pumps; breast implants;
dental implants; surgical mesh; and implantable access systems.
Examples of implantable devices also include topical devices such
as bandages, wraps and tapes that are applied to skin wounds. In
some embodiments of the skin wound, at least a section of the skin
surface layer, the epidermis, which is naturally acidic, is wounded
or damaged, such that the exposed skin inner layer or tissue has a
physiological pH of above 7 absent any microbial growth.
[0029] Alternatively, the duration of the implant may be temporary
or transient. That is to say that the implant is intended to remain
in place for a defined period of time which, however, is sufficient
to allow an infection to develop. Temporary implants may be
inserted from 1 day through 2 years or longer. Examples of
temporary implants include sutures, catheters, intravenous
injection ports, braces, and the like. Examples of transient
implantable medical devices include, without limitation, syringes
whose tip can be introduced into a patient's body or a natural
orifice and catheters. In some embodiments, the tip of the syringe
or catheter comprises indicators attached or incorporated thereto
and a removable cap. The cap covers the area of the tip having the
indicators and insulates the tip from outside environment so that
it is not contaminated with materials, such as physiological fluid
or tissue, that are not to be tested. The cap can be opened, such
as by pushing the tip, when the tip is placed at a location where
possible microbial growth is to be detected, and closed after a
sample is retrieved by the tip to protect the tip and the sample
from contamination when device is withdrawn from the location being
tested. The absence or presence of microbial growth can be
determined by detection of the signals produced by the indicators
on the tip with or without removing the cap.
[0030] The term "surface" as it relates to the implantable medical
device refers to the outer surface of the device interfacing with
physiological fluid and tissue or organs of a patient. For example,
both the exterior and the interior walls of the lumen of a catheter
are outer surfaces of the catheter as both the exterior and the
interior walls can be in contact with a physiological fluid or
tissue when used in a medical procedure. Similarly, the interior
wall of the cartridge of a syringe is an outer surface of the
syringe as the interior wall can be in contact with a physiological
fluid or tissue. As described below, in a preferred embodiment, the
surface of the medical device comprises a surface layer to which an
indicator or reporter has been integrated therein or can be
attached by post-treatment to from covalent linkages thereto. In an
embodiment, the surface comprises a mesh or similar covering, for
example the surgical mesh pouches disclosed in PCT Pub. No. WO
2008/127411. In yet another embodiment, the surface comprises a
biodegradable or bioerodable layer which covers and thereby
protects the indicators and/or reporters during implantation. In
such an embodiment, the surface of the medical device constitutes
three components--the inner component defining the surface of the
medical device; an intermediate component which comprises a
plurality of indicators and/or reporters bound to the medical
device surface; and an outer component which is a biodegradable or
bioerodable layer forming the outer surface.
[0031] The term "biodegradable or bioerodable layer" refers to a
biocompatible material which degrades or erodes in vivo to expose
the underlying surface. Such materials can be any material well
known in the art which provides for an outer coating on the device.
For example, biodegradable materials include hyaluronic acid,
collagen, polylactides, polyglycolides, polycaprolactones,
polydioxanones, polycarbonates, polyhydroxybutyrates, polyalkylene
oxalates, polyanhydrides, polyamides, polyesteramides,
polyurethanes, polyacetals, polyketals, polyorthocarbonates,
polyphosphazenes, polyhydroxyvalerates, polyalkylene succinates,
poly(malic acid), poly(amino acids), chitin, chitosan, and
polyorthoesters, and copolymers, terpolymers and combinations and
mixtures thereof. See, for example, Dunn, et al., U.S. Pat. No.
4,938,763 which is incorporated herein by reference in its
entirety.
[0032] The term "patient" refers to any mammalian patient and
includes without limitation primates such as humans, monkeys, apes,
and the like, and domesticated animals such as horses, dogs, cats,
ovines, bovines, and the like.
[0033] As used herein, the term "indicator" refers to a compound or
device that produces a signal in presence of microbial growth. The
signal can be electromagnetic such as a change in absorption which
can be observed by naked eye and/or by using an emission and/or
absorption spectrophotometer. Such indicators include by way of
example only, dyes including pH indicators, metals such as
gadolinium, pH sensitive fluorescent indicators and the like.
[0034] Suitable pH indicators include, by way of example only,
phenol red, xylenol blue, bromocresol purple, bromocresol green,
Congo red, cresol red, phenolphthalein, bromothymol blue,
p-naphtholbenzein, neutral red, a mixture of potassium iodide,
mercuric (III) iodide, sodium borate, sodium hydroxide and water
nile blue, thymolphthalein, crysol violet, hydroxy naphthol blue,
malachite green oxalate, methyl orange, alizarin, crystal violet,
methyl red, fluorescein, and derivatives and mixtures thereof as
well as food grade dyes provided that in each case such indicators
generate a signal when in the presence of microbial growth.
Suitable pH sensitive fluorescent indicators include, but not
limited to, 6,7-dihydroxy-4-methylcoumarin, 7-hydroxycoumarin and
derivatives thereof, which are non-fluorescent in acidic pH and
become blue fluorescent toward neutral pH. The structures of these
is given below:
##STR00001##
[0035] In some embodiments, the indicator described herein is a
metal selected from the group of a fluorescent moiety; a
paramagnetic ion, such as gadolinium, europium, manganese,
lanthanide, iron, and derivatives thereof; or a phase transition
material. The indicator is capable of remote detection, for example
by magnetic resonance imaging (MRI). Examples of these and other
indicators are well-known in the art. For example, and without
limiting the scope of the present invention, Amanlou, et al.
describes several indicators that are commonly used in MRI,
including small mononuclear or polynuclear paramagnetic chelates;
metalloporphyrins; polymeric or macromolecular carriers (covalently
or noncovalently labeled with paramagnetic chelates); particulate
contrast agents (including fluorinated or non-fluorinated
paramagnetic micelles or liposomes) and paramagnetic or super
paramagnetic particles (e.g., iron oxides, Gd3-labeled zeolites);
dimagnetic CEST polymers; dimagnetic hyperpolarization probes
(gases and aerosols), and 13C-labeled compounds or ions. Amanlou,
et al., Current Radiopharmaceuticals 4, 31-43 (2011).
[0036] In some embodiments, the indicator is pH-sensitive or
temperature-sensitive.
[0037] In an embodiment, the indicator is a fluorescent moiety.
Fluorescence is the light emitted when a molecule absorbs light at
a higher energy wavelength and emits that light at a lower energy
wavelength. In an embodiment, the fluorescent moiety is remotely
detectable, for example by fluorescence spectroscopy.
[0038] In an embodiment, the fluorescent moiety is present in a
liposome at self-quenching concentration. In an embodiment, a
liposome comprises the fluorescent moiety and a fluorescent
quencher.
[0039] In an embodiment, the indicator is fluorescein or a
derivative thereof, or a salt of fluorescein or the derivative.
[0040] Fluorescein is of the formula:
##STR00002##
or a tautomeric structure.
[0041] Salts of fluorescein or a derivative include, but are not
limited to, the sodium salt and disodium salt, potassium salt and
dipotassium salt.
[0042] Peak excitation of fluorescein occurs at 494 nm and peak
emission at 521 nm. The absorption and fluorescence of fluorescein
or its derivatives are pH dependent. For example, fluorescein has a
pKa of 6.4, and its ionization equilibrium leads to pH-dependent
absorption and emission over the range of 5 to 9. Extinction
coefficients and fluorescence quantum of fluorescein yields
decrease at pH<7, such as pH 5.5. The fluorescence lifetimes of
the protonated and deprotonated forms of fluorescein are
approximately 3 and 4 ns, which allows for pH determination from
nonintensity based measurements. Determination of pH according to
the absorption and emission of fluorescein is known in the art,
see, e.g. Joseph R. Lakowicz, Principles of Fluorescence
Spectroscopy, 638-639 (3rd ed. 2006).
[0043] In some embodiments, the fluorescein derivative is of the
formula:
##STR00003##
or a tautomer therefore or a pharmaceutically acceptable salt of
the compound or tautomer, wherein [0044] R.sup.1, R.sup.2, R.sup.3
and R.sup.4 are independently selected from the group consisting of
hydrogen, fluoro, chloro, bromo, and --O--C.sub.1-C.sub.4 alkyl,
[0045] R.sup.5 is hydrogen, COOH or
--(C(O)NH).sub.m(CH.sub.2)).sub.n--COOH, m is 0 or 1, n is 1, 2, 3,
4, or 5; and [0046] R.sup.10 and R.sup.11 are independently
hydrogen or --C(O)C.sub.1-C.sub.4 alkyl.
[0047] In some embodiments, R.sup.2 and R.sup.3 are hydrogen.
[0048] In some embodiments, R.sup.10 and R.sup.11 are hydrogen.
[0049] Examples of fluorescein derivatives include but are not
limited to, 2',7'-dichlorofluorescein, 5(6)-carboxyfluorescein,
5(6)-carboxyfluorescein diacetate, 5-carboxyfluorescein,
6-[fluorescein-5(6)-carboxamido]hexanoic acid,
6-carboxyfluorescein, fluorescein diacetate 5-maleimide,
fluorescein-O'-acetic acid which are available from Sigma-Aldrich
Co., Missouri, USA. Additional fluorescein derivatives include
2',7'-difluorofluorescein (OREGON GREEN.TM.)
[0050] Use of the fluorescent indicators in the detection of pH
changes is known in the art and is described in, for example,
Junyan Han and Kevin Burgess, Fluorescent Indicators for
Intracellular pH, Chem. Rev., 2010, 110(5):2709-2728, which is
incorporated herein by reference in its entirety.
[0051] In an embodiment, the indicator is sensitive to pH.
pH-sensitive indicators are described, for example, in U.S. Patent
Pub. No. 2011/0104261 A1 and references therein, all of which are
incorporated herein by reference in their entirety. Such
pH-sensitive indicators may include citraconyl-linked Gd chelates,
Gd diethylenetriamine pentaacetic acid (DTPA) chelates, and Gd-DOTA
chelates.
[0052] In a preferred embodiment, the fluorescent moiety is
heptamethoxy red (HMR). The absorbance of light by HMR in a
non-acidic environment is different compared to that of acidic HMR.
Specifically, acidic HMR has absorbance in the blue light range
whereas neutral HMR does not. See FIGS. 1 and 2 which illustrate
this differential absorption pattern. In turn, this allows for
detection of which form of the HMR exists in a given sample and, in
a physiological fluid, allows for ascertaining whether that fluid
is acidic or not as well as the degree of acidity.
[0053] In some embodiments, the fluorescent moiety and the
indicator are identical. In other embodiments, the fluorescent
moiety and the indicator are different. In addition, fluorescent pH
indicators are well known in the art and some of which are
commercially available. In a preferred embodiment, such fluorescent
pH indicators can sense pH changes within physiological ranges.
See, for example,
http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-
-Handbook/pH-Indicators/Overview-of-pH-Indicators.html
[0054] In some embodiments, the indicator is covalently bound to at
least a portion of the surface of the device. In another
embodiment, the indicator is integrated at least into or on a
portion of the surface layer of the device.
[0055] In some embodiments, the indicators utilized herein are
selected from hexamethoxy red, heptamethoxy red and derivatives
thereof. Methods of making hexamethoxy red and heptamethoxy red are
well known to the skilled artisan. See, for example, U.S. Pat. No.
8,425,996, which is incorporated herein in its entirety by
reference. Derivatives of hexamethoxy red and heptamethoxy red,
including those that are capable of covalently binding to a
compatible functional group on the surface of the medical device,
and methods of their synthesis are disclosed in U.S. patent
application Ser. No. 13/715,014, which is incorporated herein in
its entirety by reference.
[0056] Specifically, hexamethoxy red and heptamethoxy red can be
synthesized following art recognized methods with the appropriate
substitution of commercially available reagents as needed. Other
compounds are synthesized following modifications of the methods
illustrated herein, and those known, based on this disclosure. See,
for example, Raj. B. Durairaj, Resorcinol: Chemistry, Technology,
and Applications, Birkhauser, 2005. Illustrative and non-limiting
methods for synthesizing such compounds are schematically shown
below which show the synthesis of an intermediate 4-hydroxyphenyl
compound. That compound is subsequently modified on the hydroxyl
group to incorporate the polymerizable group or to attach to at
least a portion of the surface of a device.
##STR00004## ##STR00005##
[0057] In step 1, a protected resorcinol methyl ether is
brominated, preferably using 1 equivalent of bromine in a non-polar
solvent such as dioxane. As used herein, PG refers to a protecting
group, which refers to well known functional groups that, when
bound to a functional group, render the resulting protected
functional group inert to the reaction to be conducted on other
portions of the compound and the corresponding reaction condition,
and which can be reacted to regenerate the original functionality
under deprotection conditions. Examples of protecting groups useful
for synthesizing the compounds of this invention, and methods for
protection and deprotection employed herein, are found in standard
reference works such as Greene and Wuts, Protective Groups in
Organic Synthesis., 2d Ed., 1991, John Wiley & Sons, and
McOmie, Protective Groups in Organic Chemistry, 1975, Plenum Press.
Methylthiomethyl ether and allyl ethers are certain non-limiting
protecting groups contemplated for the scheme above. In step 2, the
brominated resorcinol derivative is metalated to provide a Grignard
reagent or a resorcyl lithium. In step 3, the metalated aryl is
reacted with an aryl carboxylic acid ester to provide a protected
precursor to the compound of Formula (I), which is deprotected in
step 4.
##STR00006## ##STR00007##
[0058] In step 5, the deprotected phenolic hydroxy compound is
reacted with an R.sup.9-L moiety containing a leaving group such as
chloro, bromo, iodo, or --OSO.sub.2R.sub.S where R.sub.S is
C.sub.1-C.sub.6 alkyl optionally substituted with 1-5 fluoro atoms
or aryl optionally substituted with 1-3 C.sub.1-C.sub.6 alkyl or
halo groups. Alternatively, the deprotected compound is reacted
with a compound that provides part of the linker L (step 6). Such
compounds can be elaborated as shown in steps 7 and 8 below using
reagents and methods well known to the skilled artisan.
##STR00008## ##STR00009##
[0059] These compounds are also synthesized by reacting an
appropriately protected aryl carboxylic acid ester with the
metalated aryl compound and elaborating the triaryl methyl
compounds produced, via methods provided herein and/or via methods
well known to the skilled artisan.
[0060] These compounds can also be synthesized by reacting an
appropriately protected aryl carboxylic acid ester with the
metalated aryl compound and elaborating the triaryl methyl
compounds produced, via methods provided herein and/or via methods
well known to the skilled artisan:
##STR00010##
[0061] Other compounds for use herein are conveniently synthesized
following these and other known methods upon appropriate
substitution of starting material and, if needed, protecting
groups. Electron withdrawing substituents such as halo can be
conveniently incorporated into the aryl rings by electrophilic
substitution employing hypohalite, halogens, ICl, preferably under
alkaline conditions. A halo group is conveniently converted to a
cyano group following well known methods, such as those employing
CuCN. A nitro group is conveniently incorporated by electrophilic
nitration employing various conditions and reagents well known to
the skilled artisan, such as nitronium tetrafluoroborate, nitric
acid, optionally with acetic anhydride, and the like.
[0062] Other compounds can be prepared following methods well known
to a skilled artisan and/or those disclosed herein upon appropriate
substitution of reactants and reagents.
[0063] Preferred compounds for use herein include those represented
below:
##STR00011##
TABLE-US-00001 TABLE 1 Ex. R.sup.2, No. R.sup.1 R.sup.4, R.sup.6
R.sup.3 R.sup.5 R.sup.7 1 H --CH.sub.3 --CH.sub.3 --CH.sub.3
--CH.sub.2CH.dbd.CH.sub.2 2 H --CH.sub.3 --CH.sub.2CH.dbd.CH.sub.2
--CH.sub.3 --CH.sub.3 3 H --CH.sub.3 --CH.sub.3
--CH.sub.2CH.dbd.CH.sub.2 --CH.sub.3 4 H --CH.sub.3
--CH.sub.2CH.dbd.CH.sub.2 --CH.sub.2CH.dbd.CH.sub.2 --CH.sub.3 5 H
--CH.sub.3 --CH.sub.3 --CH.sub.2CH.dbd.CH.sub.2
--CH.sub.2CH.dbd.CH.sub.2 6 H --CH.sub.3 --CH.sub.2CH.dbd.CH.sub.2
--CH.sub.3 --CH.sub.2CH.dbd.CH.sub.2 7 H --CH.sub.3
--CH.sub.2CH.dbd.CH.sub.2 --CH.sub.2CH.dbd.CH.sub.2
--CH.sub.2CH.dbd.CH.sub.2 8 H --CH.sub.3 --CH.sub.3 --CH.sub.3
--CH.sub.2CH.sub.2OC(O)CH.dbd.CH.sub.2 2-(acryl)ethylene 9 H
--CH.sub.3 --CH.sub.3 2-(acryl)ethylene --CH.sub.3 10 H --CH.sub.3
2-(acryl)ethylene 2-(acryl)ethylene --CH.sub.3 11 H --CH.sub.3
Allyl Acrylate --CH.sub.3 12 H --CH.sub.3
--(CH.sub.2).sub.nN.dbd.C.dbd.O --(CH.sub.2).sub.nN.dbd.C.dbd.S
--CH.sub.3 where n = 2-12 where n = 2-12 13 H --CH.sub.3
--(CH.sub.2).sub.nN.dbd.C.dbd.O --(CH.sub.2).sub.nN.dbd.C.dbd.O
--CH.sub.3 where n = 2-12 where n = 2-12 14 H --CH.sub.3
--(CH.sub.2).sub.nN.dbd.C.dbd.S --(CH.sub.2).sub.nN.dbd.C.dbd.S
--CH.sub.3 where n = 2-12 where n = 2-12 15 H --CH.sub.3
--(CH.sub.2).sub.nCO.sub.2Ar.sub.F
--(CH.sub.2).sub.nCO.sub.2Ar.sub.F --CH.sub.3 where Ar.sub.F is
penta where Ar.sub.F is penta or tetrafluorophenyl or
tetrafluorophenyl 16 H --CH.sub.3 --(CH.sub.2).sub.nN.sub.3 where n
= --(CH.sub.2).sub.nN.sub.3 where n = --CH.sub.3 2-12 2-12 17 H
--CH.sub.3 --(CH.sub.2).sub.nC.ident.CH where
--(CH.sub.2).sub.nC.ident.CH --CH.sub.3 n = 2-12 where n = 2-12 18
H --CH.sub.3 --(CH.sub.2).sub.nN.dbd.C.dbd.O
--(CH.sub.2).sub.nN.dbd.C.dbd.O --CH.sub.3 where n = 2-12 where n =
2-12 19 H --CH.sub.3 --CH.sub.3 --(CH.sub.2).sub.nN.dbd.C.dbd.S
--CH.sub.3 where n = 2-12 20 H --CH.sub.3 --CH.sub.3
--(CH.sub.2).sub.nCO.sub.2Ar.sub.F --CH.sub.3 where Ar.sub.F is
penta or tetrafluorophenyl 21 H --CH.sub.3 --CH.sub.3
--(CH.sub.2).sub.nN.sub.3 where n = --CH.sub.3 2-12 22 H --CH.sub.3
--CH.sub.3 --(CH.sub.2).sub.nC.ident.CH --CH.sub.3 where n = 2-12
23 --OMe --CH.sub.3 --CH.sub.3 --CH.sub.3 --CH.sub.2CH.dbd.CH.sub.2
24 --OMe --CH.sub.3 --CH.sub.2CH.dbd.CH.sub.2 --CH.sub.3 --CH.sub.3
25 --OMe --CH.sub.3 --CH.sub.3 --CH.sub.2CH.dbd.CH.sub.2 --CH.sub.3
26 --OMe --CH.sub.3 --CH.sub.2CH.dbd.CH.sub.2
--CH.sub.2CH.dbd.CH.sub.2 --CH.sub.3 27 --OMe --CH.sub.3 --CH.sub.3
--CH.sub.2CH.dbd.CH.sub.2 --CH.sub.2CH.dbd.CH.sub.2 28 --OMe
--CH.sub.3 --CH.sub.2CH.dbd.CH.sub.2 --CH.sub.3
--CH.sub.2CH.dbd.CH.sub.2 29 --OMe --CH.sub.3
--CH.sub.2CH.dbd.CH.sub.2 --CH.sub.2CH.dbd.CH.sub.2
--CH.sub.2CH.dbd.CH.sub.2 30 --OMe --CH.sub.3 --CH.sub.3 --CH.sub.3
--CH.sub.2CH.sub.2OC(O)CH.dbd.CH.sub.2 2-(acryl)ethylene 31 --OMe
--CH.sub.3 --CH.sub.3 2-(acryl)ethylene --CH.sub.3 32 --OMe
--CH.sub.3 2-(acryl)ethylene 2-(acryl)ethylene --CH.sub.3 33 --OMe
--CH.sub.3 Allyl Acrylate --CH.sub.3 34 --OMe --CH.sub.3
--(CH.sub.2).sub.nN.dbd.C.dbd.O --(CH.sub.2).sub.nN.dbd.C.dbd.S
--CH.sub.3 where n = 2-12 where n = 2-12 35 --OMe --CH.sub.3
--(CH.sub.2).sub.nN.dbd.C.dbd.O --(CH.sub.2).sub.nN.dbd.C.dbd.O
--CH.sub.3 where n = 2-12 where n = 2-12 36 --OMe --CH.sub.3
--(CH.sub.2).sub.nN.dbd.C.dbd.S --(CH.sub.2).sub.nN.dbd.C.dbd.S
--CH.sub.3 where n = 2-12 where n = 2-12 37 --OMe --CH.sub.3
--(CH.sub.2).sub.nCO.sub.2Ar.sub.F
--(CH.sub.2).sub.nCO.sub.2Ar.sub.F --CH.sub.3 where Ar.sub.F is
penta where Ar.sub.F is penta or tetrafluorophenyl or
tetrafluorophenyl 38 --OMe --CH.sub.3 --(CH.sub.2).sub.nN.sub.3
where n = --(CH.sub.2).sub.nN.sub.3 where n = --CH.sub.3 2-12 2-12
39 --OMe --CH.sub.3 --(CH.sub.2).sub.nC.ident.CH where
--(CH.sub.2).sub.nC.ident.CH --CH.sub.3 n = 2-12 where n = 2-12 40
--OMe --CH.sub.3 --(CH.sub.2).sub.nN.dbd.C.dbd.O
--(CH.sub.2).sub.nN.dbd.C.dbd.O --CH.sub.3 where n = 2-12 where n =
2-12 41 --OMe --CH.sub.3 --CH.sub.3 --(CH.sub.2).sub.nN.dbd.C.dbd.S
--CH.sub.3 where n = 2-12 42 --OMe --CH.sub.3 --CH.sub.3
--(CH.sub.2).sub.nCO.sub.2Ar.sub.F --CH.sub.3 where Ar.sub.F is
penta or tetrafluorophenyl 43 --OMe --CH.sub.3 --CH.sub.3
--(CH.sub.2).sub.nN.sub.3 where n = --CH.sub.3 2-12 44 --OMe
--CH.sub.3 --CH.sub.3 --(CH.sub.2).sub.nC.ident.CH --CH.sub.3 where
n = 2-12 45 H --CH.sub.3 --(CH.sub.2).sub.nOC(O)CH.dbd.CH.sub.2
--(CH.sub.2).sub.nOC(O)CH.dbd.CH.sub.2 --CH.sub.3 where n = 2-12
where n = 2-12 46 H --CH.sub.3 --CH.sub.3
--(CH.sub.2).sub.nOC(O)CH.dbd.CH.sub.2 --CH.sub.3 where n = 2-12 47
--OMe --CH.sub.3 --(CH.sub.2).sub.nOC(O)--CH.dbd.CH.sub.2
--(CH.sub.2).sub.nOC(O)--CH.dbd.CH.sub.2 --CH.sub.3 where n = 2-12
where n = 2-12 48 --OMe --CH.sub.3 --CH.sub.3
--(CH.sub.2).sub.nOC(O)--CH.dbd.CH.sub.2 --CH.sub.3 where n =
2-12
[0064] As is apparent, the above indicators with reactive moieties
can be utilized as a reactive monomer so as to be integrated into a
polymer matrix. In another embodiment, the reactive moieties can be
used to form a covalent bond with a compatible reactive
functionality on the polymer. For example, an isocyanate moiety can
react with an amine or hydroxyl group present on the polymer such
as poly(2-hydroxyethylmethacrylate). This post-treatment process
allows for site specific application of the indicator to designated
areas of the polymer.
[0065] Other indicators suitable for use in this invention are
those which produce a signal such as an electromagnetic signal upon
change in pH from neutral to acidic. Such indicators are well known
in the art and include, by way of example only,
TABLE-US-00002 TABLE 2 Indicator Representative structure Example
of reactive derivative Phenol red ##STR00012## ##STR00013## Xylenol
Blue ##STR00014## ##STR00015## Methyl orange ##STR00016##
##STR00017##
Such reactive compounds can be readily prepared by those skilled in
the art.
[0066] The term "pharmaceutically acceptable salt" refers to a salt
of the compound described herein that is, within the scope of sound
medical judgment, suitable for use in contact with the tissues of
subjects without undue toxicity, irritation, allergic response, and
the like, commensurate with a reasonable benefit/risk ratio, and
effective for their intended use, as well as the zwitterionic
forms, where possible, of the compounds described herein. The term
salts refers to the relatively non-toxic, inorganic and organic
acid addition salts of the compounds described herein. These salts
can be prepared in situ during the isolation and purification of
the compounds or by separately reacting the purified compound in
its free base form with a suitable organic or inorganic acid and
isolating the salt thus formed. Representative salts include the
hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate,
oxalate, valerate, oleate, palmitate, stearate, laurate, borate,
benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate,
succinate, tartrate, naphthylate mesylate, glucoheptonate,
lactobionate, methane sulphonate, and laurylsulphonate salts, and
the like. These may include cations based on the alkali and
alkaline earth metals, such as sodium, lithium, potassium, calcium,
magnesium, and the like, as well as non-toxic ammonium, quaternary
ammonium, and amine cations including, but not limited to ammonium,
tetramethylammonium, tetraethylammonium, methylamine,
dimethylamine, trimethylamine, triethylamine, ethylamine, and the
like. (See S. M. Barge et al., J. Pharm. Sci. (1977) 66, 1, which
is incorporated herein by reference in its entirety, at least, for
compositions taught therein).
[0067] The term "signal" refers to any signal that can be detected
remotely which signal correlates to the presence of active
microbial growth or infection at or adjacent to the site of
implantation of the medical device. The signal can be a color
change which can be detected by an indicator attached to the
medical device and which indicator emits information (typically in
the form of readable electromagnetic energy) which can be detected
ex vivo. Preferably, the signal is directly in the form of
electromagnetic energy which penetrates out of the body and can be
ascertained by merely monitoring for that energy.
[0068] The term "ex vivo" refers to monitoring or assessment of a
signal emitted from the indicator or reporter of the invention
located inside the body of a patient using equipment or devices
outside the body. That is to say, the signal can be monitored
without invasive procedures.
[0069] The term "detecting" refers to the use of any device which
can determine the presence of a signal. In an embodiment, the
signal is monitored continuously such that a machine-readable
signal is detected and reported on an on-going basis. In an
embodiment, the signal is detected and monitored intermittently,
for example periodically every few hours or days. In an embodiment,
the signal is detected at discrete times, for example when
infection is suspected or when the patient visits a health care
facility (e.g., routine check-ups).
[0070] The term "electromagnetic energy" refers to any wavelength
of energy capable of being transmitted from the body as well as
being monitored ex vivo. Examples of such energy include light in
the ultraviolet (UV), visible and infrared (IR) portions of the
light spectrum. Other examples include energy readable by magnetic
resonance imaging (MRI), X-rays, and the like.
[0071] The term "produce or can be induced to produce a signal"
means that the indicator directly or indirectly produces a signal.
An example of indirect production of a signal is the use of energy
directed to the indicator to induce fluorescence.
[0072] The term "blue light" refers to light that has a wavelength
of about 450-500 nm and is more energetic than red light which has
a wavelength of about 620 to 750 nm. Blue light penetrates skin
well and frequently is used to treat jaundice in newborns by
breaking down bilirubin in the blood. In this invention,
irradiation of an implanted medical device having covalently bound
thereto HMR will allow absorption of the blue light and emittance
of fluorescence if there is an active infection.
[0073] Alternatively, HMR incorporated into a pH-dependent liposome
will be released from degraded liposomes in the presence of acidic
pH. That is to say, that microbial growth or an active infection
will create an acidic microenvironment which, in turn, will degrade
the liposomes and/or alter the structure of HMR into a form that
absorbs blue light. Irradiation of the skin area when the implant
is made, such as an artificial knee, will indicate infection by
virtue of the acidic nature of the microenvironment which can be
detected non-invasively by the fluorescence emitted.
[0074] The term "pH dependent liposomes" refers to those well-known
liposomes which are stable at neutral or alkaline pH but which are
unstable under acidic pH conditions. U.S. Patent Pub. No.
2011/0104261 A1, which is incorporated herein by reference in its
entirety, discloses pH-sensitive liposomal probes. pH-degradable
compositions, including liposomes, are disclosed in U.S. Patent
Pub. No. US2013/0064772, and PCT International Patent Pub. No. WO
2013/036771, each of which is hereby incorporated by reference into
this application in its entirety.
[0075] The term "temperature-sensitive liposomes" refers to those
liposomes which are stable at normal body temperature (around
37.degree. C.) but degrade at higher temperatures, such as those
present at infection sites. Temperature-sensitive liposomes may be
comprised of, for example, dipalmitoylphosphatidylcholine (DPPC) or
natural or synthetic thermosensitive polymers. See, for example,
Kono and Takagishi, "Temperature-Sensitive Liposomes", Methods in
Enzymology 387, 73-82 (2004).
[0076] Liposomes may be comprised of any naturally-occurring or
synthetic lipids and/or lipophilic compounds, including, without
limitation, phosphatidylcholine, charged lipids (e.g.,
stearlamine), cholesterol, and/or aminoglycosides. Liposomes,
including pH-sensitive liposomes, may also include lipids,
lipophilic compounds, and pH-responsive copolymers as described in
U.S. Patent Pub. No. 2011/0104261 A1. Liposomes that are sensitive
to pH may comprise, for example, a blend of
phosphatidylethanolamine (PE), or a derivative thereof, compounds
containing an acidic group (e.g., carboxylic group) that acts as
stabilizer at neutral pH; pH-sensitive lipids; synthetic fusogenic
peptides/proteins; dioleoylphosphatidylethanolamine; and/or
attachment of pH-sensitive polymers to liposomes. Use of other
compounds, for example distearoylphosphatidylcholine, hydrogenated
soya PC, lipid conjugates, phosphatidylethanolamine-poly(ethylene
glycol), poly[N-(2-hydroxypropyl)methacrylamide)],
poly-N-vinylpyrrolidones, L-amino-acid-based biodegradable
polymer-lipid conjugates, or polyvinyl alcohol may allow for
decreased leakage of encapsulated compounds and/or longer-lasting
liposomes. Similarly, coating surface with inert biocompatible
polymers (such as polyethylene glycol, PEG), can increase the
longevity of liposomes in vivo while also allowing such polymers to
be detached by acidic pH.
[0077] In some embodiments, nanoparticles other than lipids may be
used to form a delivery vehicle analogous to a liposome. The term
"liposome" is meant to encompass such analogous structures.
[0078] The term "reporter" refers to compounds that are bound to an
antibody or binding fragment thereof and which change at least one
of their electromagnetic emission characters when bound to the
microbe as compared to that when not bound to the microbe. For
example, reporters can be fluorescent indicators which have an
altered fluorescence when bound to the microbe as compared to being
unbound. For example, the fluorescence signal may be quenched due
to the proximity of a quenching molecule in the absence of a
microbe; binding of the antibody or fragment to the microbe results
in a conformational change such that the quenching molecule is no
longer in close enough proximity to exert a quenching effect.
[0079] Antibodies, and fragments thereof, that are specific for a
variety of infectious bacteria and other microbes are well-known in
the art. For example, U.S. Pat. No. 7,531,633 B2 discloses
antibodies specific for Staphylococcus aureus. U.S. Patent
Application Pub. No. 2013/0022997 discloses antibodies specific for
methicillin-resistant Staphylococcus aureus (MRSA) that can
distinguish MRSA from methicillin-sensitive Staphylococcus aureus
(MSSA). In addition, microbe- and bacteria-specific antibodies are
commercially available from a wide variety of vendors, including,
for example, Kirkegaard & Perry Laboratories, Inc. and Santa
Cruz Biotechnology.
[0080] Antibodies (including molecules comprising, or alternatively
consisting of, antibody fragments or variants thereof) should have
a binding specificity for the microbe(s) of interest such that
false positives are avoided. In some embodiments, the antibody or
fragment thereof binds to multiple related microbes. In preferred
embodiments, the antibodies or fragments thereof specifically bind
to an antigen specific for the microbe of interest and do not
cross-react with any other antigens.
[0081] In an embodiment, the antibodies are human antigen-binding
antibody fragments and include, but are not limited to, Fab, Fab'
and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies,
disulfide-linked Fvs (sdFv) and fragments comprising either a VL or
VH domain. Antigen-binding antibody fragments, including
single-chain antibodies, may comprise the variable region(s) alone
or in combination with the entirety or a portion of the following:
hinge region, CH1, CH2, and CH3 domains. Also included in the
invention are antigen-binding fragments also comprising any
combination of variable region(s) with a hinge region, CH1, CH2,
and CH3 domains. The antibodies of the invention may be from any
animal origin including birds and mammals. Preferably, the
antibodies are human, murine (e.g., mouse and rat), donkey, ship
rabbit, goat, guinea pig, camel, horse, or chicken. As used herein,
"human" antibodies include antibodies having the amino acid
sequence of a human immunoglobulin and include antibodies isolated
from human immunoglobulin libraries or from animals transgenic for
one or more human immunoglobulin and that do not express endogenous
immunoglobulins, for example those described in U.S. Pat. No.
5,939,598 by Kucherlapati et al. An antibody can be humanized,
chimeric, recombinant, bispecific, a heteroantibody, a derivative
or variant of a polyclonal or monoclonal antibody.
[0082] The term "microbe" refers to any infectious organism,
including but not limited to a bacterium, fungus, yeast, or virus.
Such organisms are well-known in the art. Common infectious
bacteria include, but are not limited to, staphylococci,
streptococci, Enterococcus faecalis, Escherichia coli, Klebsiella
pneumoniae, Proteus mirabilis, and Pseudomonas aeruginosa.
Infections are also commonly caused by Candida and
mycobacteria.
[0083] In some embodiments, the liposome is associated with at
least one antibiotic, such as a penicillin, a cephalosporin, a
carbapenem, a polymixin, a rifamycin, a lipiarmycin, a quinolone, a
sulfonamide, a .beta.-lactam, a fluoroquinolone, a glycopeptide, a
ketolide, a lincosamide, a streptogramin, an aminoglycoside, a
macrolide, a tetracycline, a cyclic lipopeptide, a glycylcycline,
or an oxazolidinone. Antibiotics in these classes are well known in
the art. One of ordinary skill in the art would understand that
this list is not exhaustive and the use of any antibiotic is within
the scope of this invention.
[0084] In some embodiments, an anti-infective agent (for example,
an antifungal triazole or amphotericin) is associated with the
liposome. These may include carbapenems, for example meropenem or
imipenem, to broaden the therapeutic effectiveness.
[0085] In some embodiments, the indicator comprises a fluorescent
moiety, a paramagnetic ion, or a pH sensitive dye which is capable
of remote detection. In an embodiment, the indicator is covalently
attached to a medical device or to a covering or coating
thereof.
[0086] In some embodiments, the indicator is associated with one or
more liposomes. In some embodiments, the liposomes further comprise
an antibody or binding fragment thereof which specifically binds to
a microbe and produces a signal indicating the identity of the
microbe bound thereto.
[0087] In some embodiments, there is provided an antibody or
binding fragment thereof has bound thereto a fluorescent moiety
which changes its fluorescent character upon binding to the microbe
or a change in pH. Such indicators include, by way of example only,
6,7-dihydroxy-4-methylcoumarin and 7-hydroxycoumarin as disclosed
above.
[0088] In some embodiments, a plurality of different antibodies or
binding fragments thereof are bound to the device each producing a
unique signal for the microbe bound thereto.
Conjugation
[0089] As necessary or as desired, the indicator or reporter can be
conjugated to the surface of the medical device by covalent bonding
through compatible functional groups. That is to say that the
surface of the medical device contains or is modified to contain a
first reactive functional group and the indicator or reporter is
modified to contain a compatible functional group. Compatible
functional groups are those functionalities which are capable of
reacting with the first reactive functional group to form a
covalent bond. Non-limiting examples of first reactive functional
groups and functional groups compatible therewith are provided in
the table below, it being understood that the first reactive
functional group and the compatible functional groups can be
interchanged. The reactions necessary to form such covalent bonds
are well known and are described in numerous standard organic
chemistry texts.
TABLE-US-00003 First Reactive Compatible Functional Covalent
Functional Group Group bond formed Hydroxyl Isocyanate Carbamate
Hydroxyl Chloroformate Carbonate Hydroxyl Thioisocyanate
Thiocarbamate Amine Carboxylic Acid Amide Amine Isocyanate Urea
Amine Thioisocyanate Thiourea Halo Phenoxide Phenylether
Detection of Microbial Growth or Infections
[0090] Exemplary and non-limiting advantages of the implantable
medical devices provided herein include the applicability to any
type of implantable medical device. Further advantages include its
ability to identify and report the presence of microbial growth or
infection adjacent to or on a medical device implanted in a
patient. For example, the microbial growth or infection may be
detected before the patient presents with the clinical effects of
such infection.
[0091] In some embodiments, the type of infection can be indicated
by the invention. For example, in one embodiment, the medical
device has one or more antibodies, or binding fragments thereof,
associated therewith. These antibodies are specific for a given
bacteria, and when bound to that bacteria produce a unique signal
evidencing the presence of the bacteria. In other embodiments,
multiple different antibodies or binding fragments thereof can be
used, each of which produces a unique signal for the presence of a
given strain of bacteria.
[0092] In some embodiments, the precise site of microbial growth
can be indicated. For example, in one embodiment, the medical
device has two or more different indicators and/or reporters
attached or incorporated to different locations of the medical
device. The signals produced by the different indicators or
reporters in response to microbial growth or infection are
different, such as fluorescent signals having different
wavelengths, and thereby the detection of a signal can be
correlated to one of the indicators or reporters, which in turn
correlates to the location of the indicator or reporter producing
the signal. For example, indicators producing signals with
wavelength A under an acidic pH can be incorporated into the inner
wall of a catheter while different indicators producing signals
with a different wavelength B under the acidic pH can be
incorporated into the outer wall of a catheter such that detection
of signals with wavelength A indicates microbial growth inside the
catheter and detection of signals with wavelength B indicates
microbial growth outside the catheter.
Uses
[0093] The implantable medical devices of this invention, in
addition to their therapeutic functions (e.g., as a prosthetic
joint), are capable of indicating the presence of infection
adjacent to or on a medical device implanted in a patient. When an
infection is suspected, or as a routine screen to detect microbial
growth before clinical signs of infection are present, the desired
imaging technology can be used to screen the implantation site for
changes in the indicator signal. The device allows early detection
and treatment of microbial growth and infection. In some
embodiments, the medical device delivers a therapeutic composition
to the site of infection. In some embodiments, the patient is
treated with antimicrobial compositions, for example orally or
intravenously.
[0094] In an embodiment, the medical device comprises a high
concentration of indicator and/or reporter associated therewith,
such that the intensity of the indicator or reporter signal under
acidic conditions is high enough to be detectable above the
background level of signal, such as that due to chromofluors
naturally present in the body. In an embodiment, the signal at the
medical device implantation site is determined after implantation
but prior to infection. This initial signal intensity can be used
as a control for background signal and compared to later signal
levels to determine whether the signal has increased or changed,
thus indicating the presence of infection.
[0095] In another embodiment, the invention also provides for a
method of detecting an infection at the surface of a wound.
Presently, the clinical diagnosis for an infection of a wound is
predicated upon site-specific pain, heat, swelling, discharge, or
redness. Though such physiological signals hold a very low
predictive value for infection. Unsurprisingly, microbiological
analysis from a tissue biopsy is often utilized as an accurate
method of confirming an infection in a wound. But this methodology
is both invasive and time-consuming, routinely taking between 48 to
72 hours allowing the infection to develop further. The present
invention obviates these drawbacks by providing a method whereby
instant detection of the infection at the surface of a wound site
is facile, safe, and non-invasive. Apropos, the invention is
applicable in cases where redness, blood, and/or bruising would
obscure typical colorimetric technology used to indicate infection.
The present invention utilizes a pH-sensitive fluorescent indicator
for such a purpose. The indicator, in the presence of blood and
other biological fluids, produces an easily identifiable and
readily distinguishable fluorescent signal indicative of microbial
growth or an infection on the surface of a wound. This fluorescence
can be monitored ex-vivo and is unaffected by bleeding, biological
fluid contamination, inflammation, and any other biological
obstruction that may occur on the surface of the wound. The present
invention also provides early detection of microbial growth,
incipient, inapparent, silent, or subclinical infection wherein
noticeable symptoms have not developed or will not develop.
Microbial growth includes incipient, inapparent, silent, or
subclinical infection as a result of microbial growth.
[0096] As used herein, the term "surface of a wound" refers to the
interface whereupon undamaged tissue is continuous until damaged
tissue interrupts the continuum in any given area of the body. Such
tissue if not limited to the skin, but can also be ocular or any
other part of the integumentary system. It is further contemplated
that a wound can comprise more than just the area where the
uppermost layer of skin is damaged. Whereas the skin has many
layers, a wound may reside in an intermediate layer of dermis
located for instance, centimeters below the upper layer of skin.
Such injuries and wounds may not be visible to the naked eye and
yet the present invention provides for a method of detection
wherein a fluorescent signal may indicate infection in this
intermediate layer, but also such a signal is detected through the
uppermost layer of dermis. In some embodiments, the surface layer
of the skin, the epidermis, which is naturally acidic, is wounded
or damaged, exposing the inner layers of the skin under the
epidermis or even the tissue under the skin which has a
physiological pH of above 7 absent any microbial growth. Detection
of an acidic pH of the exposed inner layers of the skin or tissue
under the skin is an indication of microbial growth. In some
embodiments, the wound is due to psoriasis or accidents.
[0097] One example of assessing incipient infection in a topical
wound would be the skin closure site after surgery where the site
is closed, e.g., by sutures. In one embodiment, the sutures can
integrate a reporter molecule therein including, for example,
covalent bonding. In another embodiment, a bandage can be placed
over the closed wound and the bandages interfacing the wound can
integrate a reporter molecule. In either event, fluorescence from
the reporter molecule arising from generation of an acidic pH is
evidence of incipient infection.
[0098] In some embodiments, the indicator is dispersed in the
material of the device, or a patch that can be placed on the
device.
[0099] In some embodiments, the indicator is covalently bound to a
material of the device. For example, chemically reactive
derivatives of fluorescein, such as fluorescein isothiocyanate or
carboxyfluorescein succinimidyl ester can react with a functional
group, such as amino groups on an antibody or binding fragment
thereof, or on a polymer material described herein, or hydroxyl
groups present on cellulose (e.g., cotton fiber) or a polymer
material (e.g., polyethylene glycol), such that the fluorescent
moiety of fluorescein is covalently attached to the material, which
can be incorporated into the device. Amino groups can also be
introduced to cellulose to react with chemically reactive
derivative of fluorescein, such as fluorescein isothiocyanate. See,
e.g., Qiang Yang and Xuejun Pan, A facile approach for fabricating
fluorescent cellulose, J. Applied Polymer Science, 2010, 117(6):
3639-3644, which is incorporated by reference in its entirety. In
some aspects, polyvinyl acetate polymer or a copolymer of
polyethylene glycol with polyvinyl acetate can be functionalized by
hydrolyzing a percentage (e.g., 0.1% to 10%, such as 0.1%, 0.5%,
1%, 5% or 10%, or any range between any two of the values (end
points inclusive)) of the ester groups to an alcohol groups. The
alcohol groups can also be converted to other functionalities such
as amino groups by methods known in the art. The alcohol or amino
groups, or other functional groups may react with the chemically
reactive derivatives of fluorescein so that fluorescein moieties
are incorporated into the polymer.
[0100] The term "fluorescent moiety of fluorescein" refers to the
polycyclic chemical moiety that remains after the chemically
reactive fluorescein derivative reacts with the functional groups
on a material of the device.
[0101] In some embodiments, the fluorescent moiety of fluorescein
comprises the formula:
##STR00018##
[0102] or a tautomer therefore,
[0103] wherein [0104] R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are
independently selected from the group consisting of hydrogen,
fluoro, chloro, bromo, and --O--C.sub.1-C.sub.4 alkyl, [0105]
R.sup.10 and R.sup.11 are independently hydrogen or
--C(O)C.sub.1-C.sub.4 alkyl; and represents the point of connection
to the material on the device.
[0106] In some embodiments, the chemically reactive fluorescein
derivative is a compound of the formula:
##STR00019##
or a tautomer therefore or a salt of the compound or tautomer,
wherein [0107] R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are
independently selected from the group consisting of hydrogen,
fluoro, chloro, bromo, and --O--C.sub.1-C.sub.4 alkyl, [0108]
R.sup.5 is selected from the group consisting of C.sub.1-C.sub.4
haloalkyl (C.sub.1-C.sub.4 alkyl substituted with one, two or three
chloro, bromo or iodo), --COOR.sup.6, --NR.sup.6R.sup.7,
--NHCOR.sup.6, and --N.dbd.C.dbd.S; [0109] R is selected from the
group consisting of --OH, --O--C.sub.1-C.sub.4 alkyl, and
--NR.sup.8R.sup.9; [0110] R.sup.6 is selected from the group
consisting of C.sub.1-C.sub.4 haloalkyl, a 5- or 6-membered
saturated, unsaturated or aromatic heterocycle ring comprising
carbon atoms, one, two or three nitrogen atoms, and zero to one
oxygen atom, wherein the heterocycle ring is optionally substituted
with one or two substituent selected from the group consisting of
oxo (.dbd.O), fluoro, chloro and bromo, and --C.sub.1-C.sub.4
alkyl; [0111] R.sup.7 is hydrogen, or R.sup.6 and R.sup.7 together
with the nitrogen attached thereto form a 5- or 6-membered
saturated heterocycle ring comprising carbon atoms, one or two
nitrogen atoms, and zero to one oxygen atom; [0112] R.sup.8 and
R.sup.9 are independently hydrogen or C.sub.1-C.sub.4 alkyl, or
R.sup.8 and R.sup.9 together with the nitrogen attached thereto
form a 5- or 6-membered saturated heterocycle ring comprising
carbon atoms, one or two nitrogen atoms, and zero to one oxygen
atom, wherein the heterocycle ring is optionally substituted with a
substituent selected from C.sub.1-C.sub.4 alkyl, O--C.sub.1-C.sub.4
alkyl, OH and COOH; and [0113] R.sup.10 and R.sup.11 are
independently hydrogen or --C(O)C.sub.1-C.sub.4 alkyl.
[0114] In some embodiments, R.sup.5 is --N.dbd.C.dbd.S.
[0115] In some embodiments, R.sup.5 is
##STR00020##
[0116] Examples of chemically reactive fluorescein derivatives
include but are not limited to, 5(6)-carboxyfluorescein diacetate
N-succinimidyl ester, 5-(bromomethyl)fluorescein,
5-(iodoacetamido)fluorescein, 5-carboxy-fluorescein diacetate
N-succinimidyl ester, 5-carboxyfluorescein N-succinimidyl ester,
6-carboxy-fluorescein diacetate N-succinimidyl ester,
6-[fluorescein-5(6)-carboxamido]hexanoic acid N-hydroxysuccinimide
ester, 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein
N-hydroxysuccinimide ester, fluorescein 5(6)-isothiocyanate,
fluorescein diacetate 6-isothiocyanate, and fluorescein
isothiocyanate.
[0117] In some embodiments, R.sup.5 is C.sub.1-C.sub.4 bromoalkyl.
In some aspects, the C.sub.1-C.sub.4 bromoalkyl can be converted to
a C.sub.1-C.sub.4 hydroxyalkyl, for example via hydrolysis under
basic conditions. The OH functionality in the C.sub.1-C.sub.4
hydroxyalkyl group react with methanesulfonyl chloride (mesyl
chloride, MsCl) under basic conditions (such as in the presence of
pyridine) to provide a C.sub.1-C.sub.4 alkyl mesylate. The mesylate
functionality in the C.sub.1-C.sub.4 alkyl mesylate group can react
with a OH group in a monomer such as hydroxyethylmethacrylate
(HEMA) to form a hydroxyethylmethacrylate monomer attached with a
fluorescein molecule or a fluorescein derivative. The
hydroxyethylmethacrylate monomer attached with a fluorescein
molecule or a fluorescein derivative can polymerize with other
monomers, such as HEMA or methyl methacrylate (MMA) that do not
have a fluorescent indicator attached thereto, to form polymer
material that can be used to make the outer surface of a medical
device that is capable of indicating the presence or absence of an
infection when in contact with a physiological tissue or fluid.
Such polymer may comprise 0.1% to 10% of monomers attached with a
fluorescent indicator molecule and 90% to 99.9% of monomers that
are not attached with a fluorescent indicator molecule. For
example, the monomers attached with a fluorescent indicator
molecule may be present in 0.1%, 0.5%, 1%, 5% or 10% in the
polymer, or any range between any two of the values (end points
inclusive).
[0118] The polymer material can be made into any desirable shapes
or sizes according to its use in the medical device. For example,
the polymer may be extruded as pellets, which may be made into a
shape in accordance with its use, such as the interior wall of the
cartridge of a syringe, or a catheter.
[0119] An example for preparing polymers having monomers attached
with a fluorescent indicator molecule is illustrated in the
following scheme:
##STR00021##
EXAMPLES
Example 1
Preparation of Heptamethoxy Red in Gram Scale
Step 1: Synthesis of Methyl 2,4,6-trimethoxybenzoate
[0120] 2,4,6-trimethoxybenzoic acid (5.61 g, 26.42 mmol) was
suspended in 20 mL of methanol. Concentrated sulfuric acid (1 mL)
was added to the mixture, and the reaction heated to reflux for 24
hrs. The reaction was cooled to room temperature, and the methanol
removed in vacuo. The residues were taken up in 50 mL 5%
NaHCO.sub.3 and extracted with hexane until all the solids had
dissolved. The hexane extract was dried over anhydrous
Na.sub.2SO.sub.4, filtered, and the volatiles were removed in a
rotary evaporator to dryness to give the desired product, methyl
2,4,6-trimethoxybenzoate, as a white crystalline solid.
Step 2: Synthesis of Heptamethoxy Red
[0121] 1-bromo-2,4-dimethoxybenzene (4.23 g, 19.47 mmol) was added
to a round bottom flask, and the flask flushed with nitrogen for 10
minutes. Anhydrous ether (80 mL) was added, followed by the drop
wise addition of n-butyllithium in hexane (1.6M, 12.2 mL). The
cloudy mixture was stirred at room temperature for 10 minutes.
Methyl 2,4,6-trimethoxybenzoate (2.20 g, 9.74 mmol) was dissolved
in ether, and added drop wise to the reaction mixture. After the
addition was complete, the reaction was stirred for 3 minutes
longer. The reaction was then poured into a reparatory funnel
containing 5% NH.sub.4Cl (50 mL) and shaken until a color change
was observed. The layers were separated, and the ether layer was
dried over anhydrous Na.sub.2SO.sub.4, filtered, and the volatiles
were removed in a rotary evaporator to dryness. The crude oil was
placed in the freezer. (Crude yield 6.02 g, 132%).
Example 2
One Step Preparation of Heptamethoxy Red
[0122] Add (4.23 g, 19.47 mmol) 1-bromo-2,4-dimethoxybenzene to an
appropriately sized round bottom flask. Attach a rubber septum to
seal the flask. Insert a needle into the septum as a vent and flush
the round bottom flask with nitrogen for about 10 minutes. Add (80
mL) anhydrous ether, followed by the drop wise addition of
n-butyllithium in hexane (1.6M, 12.2 mL). Stir the cloudy mixture
for 10 minutes and keep the round bottom flask on ice. Dissolve
(2.20 g, 9.74 mmol) of methyl 2,4,6-trimethoxybenzoate in about 20
ml of anhydrous ether (more than .about.20 mL can be used if
needed), and then add this drop wise to the reaction mixture. After
the addition is complete, stir the reaction mixture for about 3
minutes longer. Pour the reaction mixture into a separatory funnel
containing 5% NH.sub.4Cl (aq) (50 mL) and shake until a color
change is observed (pale orange). Allow the layers to separate, and
dry the top ether layer with about 5 g anhydrous Na.sub.2SO.sub.4,
filter, and remove the volatiles in a rotary evaporator to dryness
at 35-40.degree. C. under 400 mbar. Place the crude oil of
heptamethoxy red (yellow-orange in color) into the freezer. Yield
is .about.3.1 g.
Example 3
Preparation of Hexamethoxy Red in Gram Scale
[0123] Add (4.23 g, 19.47 mmol) 1-bromo-2,4-dimethoxybenzene to an
appropriately sized round bottom flask. Attach a rubber septum to
seal the flask. Insert a needle into the septum as a vent and flush
the round bottom flask with nitrogen for about 10 minutes. Add
anhydrous ether (80 mL), followed by the drop wise addition of
n-butyllithium in hexane (1.6M, 12.2 mL). Stir the cloudy mixture
for 10 minutes and keep the round bottom flask on ice. Dissolve
(2.20 g, 9.74 mmol) of methyl 2,4-dimethoxybenzoate in about 20 ml
of anhydrous ether (if needed, more than about 20 ml can be used),
and then add this drop wise to the reaction mixture. After the
addition is complete, stir the reaction mixture for about 3 minutes
longer. Pour the reaction mixture into a separatory funnel
containing 5% NH.sub.4Cl (aq) (50 mL) and shake until a color
change is observed (pale orange). Allow the layers to separate, and
dry the top ether layer with about 5 g anhydrous Na.sub.2SO.sub.4,
filter, and remove the volatiles in a rotary evaporator to dryness
at 35-40.degree. C. under 400 mbar. Place the crude oil of
hexamethoxy red (yellow-orange in color) into the freezer. Yield is
about 3.1 g.
Example 4
[0124] Preparation of a Polymerizable Indicator of this
Invention
[0125] Heptamethoxy red (1 molar equivalent) is heated with an
alkyl thiol (1.2-5 molar equivalents) and sodium tertiary butoxide
(1.2-5 molar equivalents) in DMF (about 0.5-2 moles/liter with
respect to hexamethoxy red). The reaction is monitored for
disappearance of hexamethoxy red and/or formation of hydroxylated
compounds. When the reaction is substantially complete, the
reaction mixture is cooled,
Br--(CH.sub.2).sub.m--OC(O)CH.dbd.CH.sub.2, where m is 2-10
(preferably in the same molar equivalent as the thiolate), added in
situ, and the reaction mixture heated again, if necessary. The
polymerizable indicator is isolated from the reaction mixture
following aqueous work up and separated by chromatography
preferably under neutral to slightly basic conditions, such as by
employing neutral or basic alumina, or by employing a slightly
alkaline eluent such as an eluent spiked with triethyl amine.
Example 5
Detection of Fluorescence
[0126] The feasibility of fluorescence as a means to detect
incipient microbial growth is dependent upon the ability of the
excitation light to penentrate skin and tissue and the ability of
the emitted light to penetrate tissue and skin so as to be
detectable. In this example, fluorescence was measured in the
following manner. A glass surface was coated with fluorescein in a
nanogram range on the surface. A chicken breast inclusive of skin,
fat and tissue and approximately 1 to 1.5 centimeters in thickness
was layered over the glass surface. Excitation light was
continuously directed to the surface of the chicken and
fluorescence was detected emitting through the chicken breast
evidencing that both the excitation light and the emitted light
were able to traverse though skin, fat and tissue.
[0127] Although the foregoing has been described in some detail by
way of illustration and example for purposes of clarity of
understanding, one of skill in the art will appreciate that certain
changes and modifications may be practiced within the scope of the
appended claims. In addition, each reference provided herein is
incorporated by reference in its entirety to the same extent as if
each reference was individually incorporated by reference.
* * * * *
References